Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections and a leading cause of sepsis-meningitis, is driving the search for new therapeutic targets and chemical inhibitors of those targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily commensalism, reducing the risk of developing resistance. Our studies and others have demonstrated that the E. coli K capsule is a critical virulence factor in urinary tract infections. The conservation of pathways involved in the assembly and export of K capsules in UTI-causing E. coli and related capsules in other bacterial pathogens makes these pathways attractive therapeutic targets. Based on the prior development of a high-throughput screening assay with a Z'factor of 0.93, we will identify inhibitors of the biogenesis of the K1 capsule, the leading capsule type among UTI-causing E. coli. Subsequently, we will focus on a leading set of inhibitors and will perform secondary assays to determine the specificity of the inhibition, to ascertain the capsule types that are affected, and to localize the target molecule. By the end of the project, we will have a series of inhibitors of capsule biogenesis that will facilitate the molecular dissection of capsule biogenesis and may form the basis for a new class of anti-infective agents. PUBLIC HEALTH RELEVANCE: Antibiotic resistance continues to rise and is compromising treatment of E. coli infections, particularly community-acquired UTI. K capsule is an important E. coli virulence factor. The development of capsule inhibitory drugs would be an important addition to anti-infective therapies.